Graft-versus-host (GVH) alloresponses mediated by donor lymphocyte infusions (DLI) administered to established murine mixed hematopoietic chimeras eliminate normal and malignant host hematopoietic cells without causing graft-versus-host disease (GVHD). Administration of similar DLI immediately following conditioning leads to severe GVHD. More potent graft-versus-tumor (GVT) effects are achieved from delayed DLI given to mixed hematopoietic chimeras than are achieved in fully allogeneic chimeras. We have observed that GVH-reactive T cells are activated, expand, produce cytokines and adopt the "memory" phenotype when administered as DLI to established mixed allogeneic chimeras. Therefore, the lack of GVHD cannot be attributed to global suppression of the alloresponse by regulatory cells. This potent GVH reaction is largely confined to the lymphohematopoietic system, as T cells do not accumulate in GVHD target tissues. We refer to this as a lymphohematopoietic GVH reaction (LGVHR). We have obtained evidence that a similar phenomenon can occur clinically when DLI are given to patients in whom mixed chimerism is established with non-myeloablative conditioning. The ability to achieve LGVHR without GVHD across MHC barriers provides an approach to achieving powerful GVT effects against lymphohematopoietic malignancies without GVHD. In order to identify the mechanisms whereby GVHR are confined to the lymphohematopoietic system following DLI, we will: 1) Compare the kinetics of GVH-reactive CD4 and CDS T cell activation, proliferation, differentiation, tissue accumulation and death in mice receiving DLI immediately following irradiation or with a delay following establishment of mixed allogeneic chimerism; we will address the significance and mechanisms of increased apoptosis and other differences in mixed chimeras vs freshly irradiated mice. 2) Compare the survival, proliferation, migratory properties and GVHD effector function of effector/memory T cells generated in a non-inflammatory environment (i.e. following DLI administration to established mixed chimeras) versus a pro-inflammatory environment (i.e. following DLI administration to freshly irradiated mice) when the cells are adoptively transferred to either type of environment. These studies will determine the extent to which T cell-intrinsic versus extrinsic environmental factors determine the capacity of a given effector/memory cell population to induce GVHD; 3) Examine the role of regulatory cell populations and of T cell homeostatic proliferation in modulating susceptibility to GVHD. The studies will synergize with those in Projects 2 and 3, and will make extensive use of Cores A, B, and C. An improved understanding of the mechanisms of LGVHR in delayed DLI recipients will advance the studies in Project 3 and ultimately the clinical use of this approach to separating GVHD and GVT effects in HLA-mismatched hematopoietic cell transplantation.